Recombinant protein production is well known in the art. Various proteins have been expressed in a variety of host cells. Protein production in filamentous fungi may be enhanced by fusing the desired protein to the signal sequence and/or a secreted polypeptide or portion thereof normally secreted from the host cell. Under most circumstances this will require further processing to release the desired protein from the fusion construct. The separation and purification of a fully cleaved protein of interest from its fusion analog in fermentor broth is a crucial and challenging component of the fungal expression of fusion proteins.
Recently, the production of monoclonal antibodies in filamentous fungi as fusion polypeptides was demonstrated. See co-pending applications U.S. Ser. No. 60/373,889 filed Apr. 18, 2002 and U.S. Ser. No. 60/411,540 filed Sep. 18, 2002, both entitled “DNA Sequences, Vectors, and Fusion Polypeptides for Secretion of Antibodies in Filamentous Fungi”, by Ward et al.
The production of the immunoglobulins is as a mixture of fusion proteins and mature (i.e., non-fused) immunoglobulins. This is an unsatisfactory composition for therapeutic purposes. Thus, it is necessary to purify not only the full-length antibodies from the host proteins but also from the various fusion proteins of the mixture. The presence of fusion proteins is an additional challenge for the purification of immunoglobulins from the Aspergillus preparation.
A vast amount of literature describing antibody purification has been reported. Liquid chromatography is by far the most commonly used, with two or more chromatographic steps mandatory for therapeutic applications. For example, it has been reported that Protein A chromatography used extensively in the recovery of immunoglobulins from a variety of sources, including cell cultures (Bioseparation and Bioprocessing: A Handbook. Volume 1, chapter 12. Publishers Wiley-VCH, 1998).
Hydrophobic Charge Induction Chromatography (HCIC) has been reported to separate proteins in a one-step elution process. Unfortunately, under current production methods that utilize fusion proteins as described above, the one-step process fails to separate the protein from its fusion analog.
For therapeutic compositions, the challenge is to come up with a rational combination of technologies to remove all relevant impurities from the antibody preparation. Thus, it would be advantageous to have a method of purifying the antibodies, antibody fragments and their respective fusion proteins. In addition, the methods described herein find use for the separation of a protein from its fusion analog.